ABSTRACT Traditional vaccines have relied upon production of antibodies to surface-exposed antigens to clear a pathogen from the body and prevent infection, yet this approach has proven ineffective against intracellular bacteria, parasites, and certain viruses. Organisms that infect a cell and replicate intracellularly are protected from antibodies and can only be cleared by cytotoxic T cells (CTLs) that target the infected cell and destroy it. A CTL response has also shown to be beneficial in treating cancer. This is particularly clear in the field of immuno-oncology where checkpoint inhibitors are revolutionizing the field by removing the ?off switch? from T cells and enabling them to effectively fight multiple cancer types. Although the need for treatments that produce a robust CTL response is clear, previous efforts have failed due to the inability to deliver the antigen to the cytosol, a requirement for a robust CTL response. Antigen delivery alternatives, including electroporation and nanoparticle-based systems, can often result in toxicity, off-target effects, low efficiency and/or accumulation of material in endosomal compartments. Furthermore, these processes are not amenable to scalable deployment, limiting the number of patients able to be treated. Thus, to develop the next generation of CTL driven therapies, the field requires new technologies capable of versatile, efficient and non-toxic delivery of antigens. In Phase II, SQZ Biotechnologies proposes to develop our vector-free microfluidic platform to deliver antigens with high efficiency directly into the cytosol of dendritic cells that can then be used for the treatment of infectious diseases and cancer. The principle underlying this approach is temporary membrane disruption by rapid mechanical deformation, or ?squeezing?, to facilitate cytosolic delivery of antigens. In SBIR Phase I, we demonstrated the feasibility of squeezing immune cells to deliver antigens and showed that with this process, we observe CTL responses that are more effective than current methods. Through cytosolic antigen delivery, we can overcome key limitations related to scalability and efficacy. In Phase II, we will (AIM 1) measure antigen-specific CTL activation and proliferation using SQZed mouse and human dendritic cells, (AIM 2) measure efficacy of antigen- specific cytotoxic T cell responses generated in vivo through tumor and infectious disease models and (AIM 3) determine if cell lysate is a potential antigen source to elicit a CTL response. Together this data will drive commercialization and partnerships for the clinical development of our novel squeezing platform.